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Mol Ther Methods Clin Dev. 2019 Nov 26;17:107-121. doi: 10.1016/j.omtm.2019.11.014. eCollection 2020 Jun 12.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

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Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA 02114, USA.
INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.
Ocular Genomics Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
The Broad Institute of Harvard and MIT, Cambridge, MA, USA.
Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.


Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.


aav; adeno-associated virus; barcode; capsid; capsid mosaics; cross-packaging; library; mosaic; viral genome

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